The use of antibodies as diagnostic tools and therapeutic modalities has found increasing use in recent years. The first FDA-approved monoclonal antibody, Rituxan® (Rituximab) was approved in 1997 for the treatment of patients with non-Hodgkin's lymphoma and soon thereafter 1998, Herceptin®, a humanized monoclonal antibody for treatment of patients with metastatic breast cancer was also approved. Numerous antibody-based therapies are showing promise in various stages of clinical development. One limitation in widespread clinical application of antibody technology is that typically large amounts of antibody are required for therapeutic efficacy and the costs associated with sufficient production are significant. Chinese Hamster Ovarian (CHO) cells and NSO2 myeloma cells are the most commonly used mammalian cell lines for commercial scale production of glycosylated human proteins like antibodies. Mammalian cell line production yields typically range from 50-250 mg/L for 5-7 day culture in a batch fermentor or 300-600 mg/L in 7-12 days in fed batch fermentors. Non-glycosylated proteins can be successfully produced in yeast (e.g., insulin production by Novo Nordisk) or E. Coli (e.g., insulin production by Eli Lilly, and Fab production by Celltech).
Previous attempts to express a full length antibody/immunoglobulin molecule via recombinant DNA technology using a single vector have met with limited success, typically resulting in unequal levels of expression of the heavy and light chains of the antibody/immunoglobulin molecule, and more particularly, a lower level of expression for the second gene. The unequal expression of heavy and light chains within the cell results in an overall low yield of full length antibody. In order to express high levels of a fully biological functional antibody from a single vector, equimolar expression of the heavy and light chains is required. Additionally, conventional vectors relying on dual promoter regulation of gene expression are invariably affected by promoter interaction (i.e., promoter interference) which may compromise equimolar expression of the genes. Other factors that limit the ability to express two or more coding sequences from a single vector include the packaging limitation of the vector itself. For example, in considering the appropriate vector/coding sequence, factors to be considered include:packaging capacity of the vector (e.g., approx. 4,500 bp for AAV), which can limit the size of expressible coding sequences; the duration of in vitro/in vivo expression of the recombinant protein by a vector-transfected cell or organ (e.g., short term expression for adenoviral vectors); the cell types infected by the vector if a viral vector is used; and the desired expression level of the gene product(s) which is generated. The requirement for controlled expression of two or more gene products together with the packaging limitations of viral vectors such as adenovirus and AAV, limits the choices with respect to vector construction and systems for expression of immunoglobulins or fragments thereof.
In order to express two or more protein or polypeptide sequences from a single vector, two or more promoters or an internal ribosome entry site (IRES) sequence are used to drive expression of individual genes. The use of two promoters within a single vector can result in low protein expression due to promoter interference. When two genes are linked with an IRES sequence, the expression level of the second gene is often significantly weaker than the first gene (Furler et al., Gene Therapy 8:864-873, 2001).
The linking of proteins in the form of polyproteins in a single open reading frame is a strategy adopted in the replication of many viruses including picomaviridae. Upon translation, virus-encoded proteinases mediate rapid intramolecular (cis) cleavage of a polyprotein to yield discrete mature protein products. Foot and Mouth Disease viruses (FMDV) are a group within the picomaviridae which express a single, long open reading frame encoding a polyprotein of approximately 225 kD. The full length translation product undergoes rapid intramolecular (cis) cleavage at the C-terminus of a 2A region occurring between the capsid protein precursor (P1-2A) and replicative domains of the polyprotein 2BC and P3, and this cleavage is mediated by proteinase-like activity of the 2A region itself (Ryan et al., J. Gen. Virol. 72:2727-2732, 1991); Vakharia et al., J. Virol. 61:3199-3207, 1987). Ryan designed constructs identifying the essential amino acid residues for expression of the cleavage activity by the FMDV 2A region. 2A domains have also been characterized from aphthoviridea and cardioviridae of the picomavirus family (Donnelly et al., J. Gen. Virol. 78:13-21, 1997).
There remains a need for improved gene expression systems for expression of full length immunoglobulins and fragments thereof which provide advantages relative to currently available technology (i.e., the use of an IRES or two or more promoters).
The present invention addresses this need by demonstrating the feasibility and use of a single vector construct which encodes a self-processing peptide for expression of a biologically functional polypeptide, such as an immunoglobulin or fragment thereof.